Detection Of Alkaline Phosphatase Research Articles

Plasmonic Enhanced Gold Nanoclusters-Based Photoelectrochemical Biosensor for Sensitive Alkaline Phosphatase Activity Analysis.

Low-toxicity gold nanoclusters-decorated Ag@SiO2 (Au NCs-Ag@SiO2) nanocomposites modified plasmonic photoelectrodes were first fabricated to improve the photoelectric properties of Au NCs and practical application in biological detection. Through adjusting distance between Au NCs and plasmonic silver nanoparticles (Ag NPs), the photocurrent intensity of Au NCs enhanced by 3.8 times attributed to strong competition between enhancement functions of hot electron transfer, local electric field, light scattering effects, and quenching functions of nonradiative energy transfer. Further comparison between experimental results and theoretical simulations were conducted to gain a deeper understanding toward the photoelectric enhancement mechanism. Moreover, Au NCs-Ag@SiO2 nanocomposites was successfully applied to the construction of photoelectrochemical (PEC) biosensors for sensitively detecting alkaline phosphatase activity. This proposed PEC biosensor showed a wide linear range from 0.04 to 400 U·L-1, and a low detection limit of 0.022 U·L-1.

Gold Nanocluster-catalyzed Luminol Chemiluminescent Sensing Method for Sensitive and Selective Detection of Alkaline Phosphatase.

A sensitive sensing method was developed for the determination of alkaline phosphatase (ALP) activity based on gold nanocluster (Au NC)-catalyzed luminol-H2O2 chemiluminescent (CL) reaction. The CL signal of luminol-H2O2-Au NCs can be quenched by ascorbic acid, which was the product of magnesium ascorbyl phosphate (MAP) hydrolysis reaction catalyzed by ALP. The proposed sensing platform showed convenient, sensitive and selective detection of ALP in the range of 0.0027 - 1.3890 U L-1, with the detection limit of 0.0026 U L-1. The broad detection linear range and ultra-high sensitivity were inherited from the efficient free radical scavenging capability of ascorbic acid on the luminol-H2O2-Au NCs CL reaction. The CL sensing platform was applied to the detection of ALP activity in serum samples. We believe that this sensing platform is a universal CL strategy for ALP detection because ascorbic acid is an efficient CL quencher for many CL reactions.

Application of apparent diffusion coefficient combined with serum tumor markers detection in evaluating neoadjuvant chemotherapy for osteosarcoma

Objective: To discuss application of apparent diffusion coefficient (ADC) in diffusion weighted imaging (DWI) combined with serum alkaline phosphatase (ALP) and tumor specific growth factor (TSGF) in evaluating neoadjuvant chemotherapy for osteosarcoma. Methods: A total of 78 patients with osteosarcoma who were admitted to People's Hospital of Gansu from January 2016 to August 2018 were collected as study subjects. All the patients were treated with neoadjuvant chemotherapy. Before chemotherapy, at the end of 4 courses of chemotherapy, before and after surgery, MRI examination and detection of serum ALP and TSGF were performed. According to results of pathological examination, the 78 patients were divided into effective chemotherapy group (n=54) and ineffective chemotherapy group (n=24). ADC values, levels of serum ALP and TSGF, change rates of ADC values and levels of serum ALP and TSGF were compared between the two groups. The value of ADC value combined with serum ALP and TSGF in evaluating curative effect of neoadjuvant chemotherapy for osteosarcoma was analyzed with receiver operating characteristic curve (ROC). Results: After chemotherapy, ADC value in effective chemotherapy group increased significantly, while levels of serum ALP and TSGF decreased significantly (t=7.269, 18.778, 23.237, all P<0.05). Only after surgery, ADC value, levels of serum ALP and TSGF increased or decreased significantly in ineffective chemotherapy group (t=7.316, 15.083, 20.930, all P<0.05). Before and after chemotherapy, change rates of ADC values and levels of serum ALP and TSGF in effective chemotherapy group were all significantly higher than those in ineffective chemotherapy group (t=7.604, 5.482, 5.048, all P<0.05). ROC curve analysis showed that area under the curve (AUC) of ADC value combined with serum ALP and TSGF for evaluating curative effect of neoadjuvant chemotherapy was 0.912, which was higher than that of ADC value, ALP, TSGF, ADC value combined with ALP, ADC value combined with TSGF (0.847, 0.787, 0.701, 0.885, 0.876, respectively). Conclusion: ADC value combined with serum tumor markers ALP and TSGF is reliable in evaluating curative effect of neoadjuvant chemotherapy for osteosarcoma.

Detection of Bacterial Alkaline Phosphatase Activity by Enzymatic In Situ Self-Assembly of the AIEgen-Peptide Conjugate.

Abnormal levels of alkaline phosphatase (ALP) activity are associated with various diseases, and many ALP probes have been developed to date. However, the development of ALP-sensitive probes for living cells, especially for the detection of bacterial ALP, remains challenging because of the complex and dynamic context. In this study, we constructed the first fluorescent probe (TPEPy-pY) for sensing bacterial ALP activity. TPEPy-pY is an AIEgen-peptide conjugate with property of aggregation-induced emission (AIE) and could turn on its fluorescence by ALP-catalyzed in situ self-assembly of the probe. The probe shows excellent selectivity and sensitivity for ALP activity, with a detection limit of 6.6 × 10-3 U mL-1. TPEPy-pY performs well in detection and in situ imaging of bacterial ALP activity against E. coli. Also, the detection does not require tedious washing steps and takes approximately 1 h, which is advantageous over commercial ALP kits. Therefore, the proposed strategy paved a new avenue for bacterial ALP detection, and we envision that more self-assembling fluorescent probes will be designed with higher sensitivity in the near future.

Synthesis of water soluble CuGaS2/ZnS quantum dots for ultrasensitive fluorescent detection of alkaline phosphatase based on inner filter effect

Developing monitoring technique for alkaline phosphatase (ALP) is crucial due to the important role it plays in living cells. Here, a kind of biocompatible glutathione-modified CuGaS2/ZnS quantum dots (GSH-CGS/ZnS QDs) was used as a fluorescent substance and then fabricated “turn-off” fluorescent biosensor for detection of ALP by help of inner filter effect (IFE). Firstly, we prepared CuGaS2/ZnS (CGS/ZnS) QDs using solvothermal method and explored the efficient ligand (GSH) exchanges strategy for transferring oil-soluble CGS/ZnS QDs to aqueous phase. More importantly, we also explored the potential biological applications of the nanohybrid QDs. The obtained GSH-CGS/ZnS QDs emitted strong yellow fluorescence with the maximum excitation (400 nm) and emission (601 nm). Then, GSH-CGS/ZnS QDs were mixed with p-nitrophenylphosphate (PNPP) and ALP. PNPP could be hydrolyzed to p-nitrophenol (PNP) by help of catalysis of ALP, and the excitation spectrum of the GSH-CGS/ZnS QDs overlapped well with the absorption spectrum of PNP, so the fluorescence of GSH-CGS/ZnS QDs was initially quenched via the so-called “IFE”. Finally, a novel “turn-off” biosensor for sensitive detection of ALP in the range of 0.05–10 U L −1(R2 = 0.98) with a detection limit of 0.01 U L−1 was successfully obtained. Results indicated that I-III-VI2 nanocrystals have great potential for their promising biomedical application.

Determining Alkaline Phosphatase Based on Core–Shell Gold@silver Nanocubes by Single-Particle Dark-Field Images

We develop a rapid and ultrasensitive colorimetric method for alkaline phosphatase (ALP) activity based on dark-field microscopy. In the presence of ALP, l-ascorbic acid 2-phosphate (AA2P) was hydrolyzed by ALP into ascorbic acid (AA), which acted as a reductant to reduce KIO3 into I2 which subsequently etched core–shell Au@Ag nanocubes. The change in morphology of the Au@Ag nanocubes led to a significant color variation (blue → yellow/green) in dark-field imaging. A linear range of 1.5 μU/mL–150 U/mL (R2 = 0.98) and limit of detection (LOD) of 0.183 μU/mL for ALP were obtained. What’s more, the successful detection of ALP in serum samples demonstrated that this sensing strategy is suitable for the ALP determination in complex biological samples.

Physical Properties, Antimicrobial Activity and In Vivo Tissue Response to Apexit Plus

We investigated the physical properties, antimicrobial activity, and tissue reaction to Apexit Plus in comparison to Sealapex. Flow, radiopacity, setting time, and solubility were evaluated in each material. The antimicrobial activity against Enterococcus faecalis was performed. Polyethylene tubes containing Apexit Plus or Sealapex, and without material (control group) were implanted into the subcutaneous tissue of rats. At 7, 15, 30, and 60 days of implantation, the specimens were paraffin-embedded and the number of inflammatory cells (ICs) and the amount of birefringent collagen (BC) were quantified. The von Kossa reaction followed by immunohistochemistry for detection of alkaline phosphatase (ALP) was also performed. Statistical analysis was performed with ANOVA and Tukey test (p ≤ 0.05). The flow value of Apexit Plus was greater than Sealapex, whereas the radiopacity (3.44 mm Al) was lower than Sealapex (6.82 mm Al). Apexit Plus showed lower solubility and shorter initial and final setting (p < 0.0001), whereas the antimicrobial activity was significantly greater than Sealapex. Although the number of ICs was higher in Apexit Plus (p = 0.0009) at 7 days, no significant difference was detected between Apexit Plus and Sealapex at 15, 30, and 60 days. All groups showed higher values for BC in the capsules over time. ALP-immunolabelled cells were observed, mainly around von Kossa-positive structures, either in the capsules of Apexit Plus or Sealapex. Therefore, our results revealed that Apexit Plus exhibited a greater effectiveness against Enterococcus faecalis and better physical properties than Sealapex, except for the radiopacity. In vivo findings indicate that Apexit Plus is biocompatible and presents potential bioactivity in the subcutaneous tissue.

A portable photoacoustic device for facile and sensitive detection of serum alkaline phosphatase activity

It is still a high challenge to develop a simple, sensitive and portable approach for bioassay in strong scattering medium. Herein, a photoacoustic (PA) device is developed for the detection of alkaline phosphatase (ALP) in serum with silver nanoparticles (AgNPs) as signal probe, without any requirements for expensive equipment, professional operation and pre-processing of real samples. ALP as an important disease marker could catalyze the breakdown of sodium L-ascorbyl-2-phosphate (AAP) into ascorbic acid (AA), thereby reducing Ag+ to AgNPs. AgNPs could generate strong PA signal under the irradiation of modulated 638-nm laser due to their localized plasmon resonance, and detected by the self-made portable PA device. Under the optimized experimental conditions, the present PA device exhibits excellent photostability and reproducibility with the relative standard deviation (RSD) of 2.2% at the concentration of 25 U L−1 ALP. Linear calibration graph is obtained within 5–70 U L−1 for ALP, along with a detection limit of 1.1 U L−1. This portable PA device is applied to detect ALP in serum samples, providing satisfactory spiking recoveries and competitive analytical performances with the current techniques. The PA-based analytical strategy obviously opens up a new avenue to the detection of disease-correlated biomarker in practice.

Developing a Cost-Effective Bioassay to Detect Alkaline Phosphatase Activity and Generating White Light Emission from a Single Nano-Assembly by Conjugating Vitamin B6 Cofactors with Lysozyme-Stabilized Fluorescent Gold Nanoclusters

In this work, lysozyme-stabilized fluorescent gold nanoclusters (Lyso-AuNCs) and pyridoxal 5′-phosphate (PLP) as a monophosphate ester substrate were used to develop a highly selective and cost-eff...

A signal amplification strategy for prostate specific antigen detection via releasing oxidase-mimics from coordination nanoparticles by alkaline phosphatase

A novel signal amplification method for prostate specific antigen (PSA) is developed by freeing fluorescein with photoinduced oxidase-like activity from coordination nanoparticles (CNPs) in the presence of alkaline phosphatase (ALP). CNPs loaded with fluorescein (F@CNPs) are obtained in aqueous solution by self-assembly using Tb3+ as metal ion, guanosine monophosphate (5′-GMP) as ligand, and fluorescein as signal molecule. The F@CNPs display outstanding properties of simple synthesis, low cost, good water solubility, negligible leakage and satisfactory load capacity. Fluorescein is quantitatively encapsulated in CNPs with a binding ratio of 92.72%. Meanwhile, ALP can specifically hydrolyze the phosphate group of 5′-GMP ligand, triggering the destruction of F@CNPs and leakage of fluorescein. Fluorescein, a photoinduced oxidase mimic, can catalyze the oxidation of non-fluorescent Amplex UltraRed (AUR) into fluorescent resorufin under LED lamp. This strategy exhibits good sensitivity for ALP detection. In addition, a new immunoassay for PSA is validated by labelling ALP on PSA antibody. The low detection limit of 0.04 ng mL−1 in detecting PSA is appropriate for PSA detection in real samples. Therefore, the work not only establishes a new strategy for ALP and PSA determination, but also provides a new conception for putting photoinduced oxidase-like fluorescein in practical application.

Enzyme-responsive Fluorescent Ionic Liquid.

Herein, we describe the development of a novel material, "enzyme-responsive fluorescent ionic liquid", which enabled a highly sensitive detection of alkaline phosphatase (ALP). We prepared a plasticized poly(vinyl chloride) (PVC) membrane using this new material as a plasticizer and quantified ALP in aqueous solutions. Preliminary results suggested that the PVC membrane responded to ALP at an interface between the membrane and the sample solution with anion extraction to maintain electroneutrality in the membrane phase. The developed PVC membrane showed an approximately six-times higher sensitivity than the conventional membrane, thereby demonstrating highly sensitive ALP detection. These results suggested the potential applicability of the proposed membrane for highly sensitive protein detection by using ALP-labeled antibodies.

Enzyme cascade reaction-based ratiometric fluorescence probe for visual monitoring the activity of alkaline phosphatase

Inspired by the intrinsic catalytic properties of alkaline phosphatase (ALP) and tyrosinase (TYR) as well as the fluorogenic reaction between levodopa and resorcinol, a ratiometric fluorescent probe based on the CdTe/CdS/ZnS/SiO2 QDs for visual monitoring the activity of ALP was developed. Phosphotyrosine was catalyzed into tyrosine by ALP, and then tyrosine was catalyzed into levodopa by TYR. Carboxyazamonardine, the reactive product of levodopa and resorcinol, gave rise to a strong blue fluorescence at 470 nm and quenched the red fluorescence of CdTe/CdS/ZnS/SiO2 QDs at 620 nm due to their inner filter effect. Thus, the resulting ratiometric fluorescence acted as the signal output for quantitative ALP detection, accompanying with the fluorescence color changing from red to purple and finally blue for semiquantitative ALP detection. A good linear relationship was obtained ranging from 0.08 to 500 U L−1 with the detection limit of 0.02 U L−1. Moreover, the probe was successfully applied to monitoring ALP activity in human serum samples without any pretreatment, and also used to screen ALP inhibitors. These results suggested that the developed method offered a highly sensitive and promising analytical platform for ALP in clinical diagnostics and paved a new way for monitoring enzymes activity visually for point-of-care diagnosis.

O-Phenylenediamine/gold nanocluster-based nanoplatform for ratiometric fluorescence detection of alkaline phosphatase activity

This study demonstrates a novel and convenient ratiometric fluorescent method for the detection of alkaline phosphatase (ALP) activity. Amino-functionalized mesoporous silica nanoparticle-gold nanoclusters (MSN-AuNCs) nanocomposites were integrated with o-phenylenediamine (OPD) to form a ratiometric fluorescence nanoplatform. The presence of ALP induced the generation of quinoxaline (QX) derivative which called 3-(dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (DFQ) with strong fluorescence emission at 450 nm, while the orange-red fluorescence of MSN-AuNCs at 580 nm was slightly quenched. Meanwhile, an obvious fluorescence color change from orange-red to purple and finally to blue can be observed by naked eyes with the increasing of ALP concentration. Therefore, employing the fluorescence emission of DFQ at 450 nm as the reporter signal and the fluorescence emission of MSN-AuNCs at 580 nm as a reference signal, a sensitive ratiometric detection method for ALP was developed. Quantitative detection of ALP activity in the linear range from 0.2 to 80 U/L with a detection limit of 0.1 U/L can be realized in this way, which endows the assay with high sensitivity enough for practical detection of ALP in human serum samples.

Amplification Strategy of Silver Nanoclusters with a Satellite-Nanostructure for Substrate-Free Assay of Alkaline Phosphatase by ICP-MS.

Alkaline phosphatase (ALP) plays critical roles in signal transmission and cell growth/apoptosis. Its abnormal level in serum/cell is tightly related to diseases, thus, serum and cellular ALP detection is of great significance for disease diagnosis. Herein, a novel approach for ALP assay based on a satellite-nanostructure is developed by conjugating lanthanide upconversion nanoparticles (UCNPs) with silver nanoclusters (AgNCs) through DNA bridging. UCNPs serve as the cores to conjugate with DNA fragments, followed by assembly of AgNCs as the satellites on UCNPs surface through the AgNCs-cytosine affinity, to produce the satellite-nanostructure of UCNPs@DNA-AgNCs. The presence of ALP converts phosphate groups into hydroxyl groups at DNA helix, weakening the coordination of DNA with UCNPs. As a result, the satellite AgNC labeling on DNA fragments strips off the UCNP surface. Silver is quantified by measuring isotope 107Ag with ICP-MS, which further derives the content of ALP by correlation to the number of AgNCs. A linear calibration range is obtained in 0.005-120 U/L with a detection limit of 1.8 mU/L. The distinct advantage of this strategy, on one hand, is the substrate-free feature that eliminates the intermediate process of substrate reaction, where the substrate activity decrease and its instability may significantly deteriorate the sensitivity. On the other hand, ALP triggers the production of a large number of AgNCs resulting in substantial amplification on ICP-MS signal to give a favorable sensitivity. This is the first attempt for ALP detection by inductively coupled plasma mass spectrometry.

Redox-modulated colorimetric detection of ascorbic acid and alkaline phosphatase activity with gold nanoparticles.

Gold nanoparticles (AuNPs) exhibit characteristic absorption peaks in the ultraviolet visible region due to their special surface plasmon resonance effect. This characteristic absorption peak would change with the relative colour varying from wine red to orange-yellow upon sequential addition of ascorbic acid (AA) into the mixture of AuNPs and Ag(I). Similar observations also could be found when the hydrolysis product of sodium l-ascorbyl-2-phosphate with alkaline phosphatase (ALP) was used as an alternative to AA. Results of structure characterization confirmed that the phenomena were due to the reduction of Ag(I) to Ag(0) on the surface of AuNPs and the formation of core-shell AuNPs@Ag. Therefore, a colorimetric assay for rapid visual detection of AA and ALP based on redox-modulated silver deposition on AuNPs has been proposed. Under the optimal experimental conditions, the absorbance variation ΔA522 nm /A370 nm of AuNPs was proportional to the concentration of AA (5-60 μmol/L) and ALP (3-18 U/L) with the corresponding detection limit of 2.44 μmol/L for AA and 0.52 U/L for ALP. The assay showed excellent selectivity towards AA and ALP. Moreover, the assay has been applied to detect AA and ALP activity in real samples with satisfying results.

Multi-emitting fluorescence sensor of MnO2–OPD–QD for the multiplex and visual detection of ascorbic acid and alkaline phosphatase

A blue-, yellow-, and red-emitting fluorescence sensor of MnO2–OPD–QD for the multiplex and visual detection of ascorbic acid and alkaline phosphatase.

Fluorescent nanosensor for in situ detection of phosphate and alkaline phosphatase in mice with parathyroid dysfunction.

A composite nanosensor based on Zr(iv)-MOFs and PNPP was developed, and was successfully applied for the in situ fluorescence imaging of phosphate and ALP levels in mice with parathyroid dysfunction. The current work provides new ideas for further development of the diagnosis of parathyroid diseases.

A ratiometric fluorescence sensor based on enzymatically activatable micellization of TPE derivatives for quantitative detection of alkaline phosphatase activity in serum.

A novel ratiometric fluorescence assay via enzymatically activatable micellization in aqueous solution was devised for quantitative detection of alkaline phosphatase (ALP) activity. We demonstrated that the dephosphorylation of the water-soluble, phosphate-functionalized, fluorophore monomer P-TPE-TG, induced by an enzymatic reaction of ALP, leads to micelle formation in aqueous solution because its water-soluble functionality is reduced. The dephosphorylation-induced micellization of P-TPE-TG exhibited a ratiometric sensing response for various ALP concentrations (10–200 mU mL−1) and provided a suitable sensing platform for naked eye detection with increased fluorescence quantum yield (Φ = 3.2%), even compared to a typical TPE-based sensor (Φ = 1.0%), where ALP can be sensed with a detection limit of 0.034 mU mL−1. In addition, P-TPE-TG displayed excellent sensing performance at concentrations from 0 to 50 mU mL−1 in diluted human serum (10%), which offers the capability to exploit ratiometric responses for bioactive substances under practical conditions.

Phosphate-responsive 2D-metal-organic-framework-nanozymes for colorimetric detection of alkaline phosphatase.

In this study, a simple colorimetric method with a tunable dynamic range for alkaline phosphatase (ALP) activity assay was developed by using a peroxidase-mimicking two-dimensional-metal-organic-framework (2D-MOF). Phosphates including pyrophosphate (PPi), ATP, and ADP inhibited the peroxidase-like activity of the 2D-MOF, while their hydrolytic product, phosphate (Pi), exhibited no such inhibitory ability. Therefore, by integrating with the ALP catalyzed hydrolytic reaction, a colorimetric method was developed for ALP activity quantification. Furthermore, by using different phosphates as inhibitors for the peroxidase-mimicking 2D-MOF, the dynamic range of this colorimetric method could be efficiently modulated. Three linear ranges of 2.5-20 U L-1, 5-60 U L-1, and 50-200 U L-1 could be obtained by using PPi, ATP, and ADP as inhibitors, respectively. Other proteins exhibited negligible interference, demonstrating that the proposed sensing method possessed excellent selectivity for ALP. Moreover, the developed assay was applied to evaluate ALP inhibitors and construct logic gates. This work not only provides a promising method for ALP detection but also represents a major step towards nanozyme bioanalysis.

A simple thermometer-based photothermometric assay for alkaline phosphatase activity based on target-induced nanoprobe generation

Alkaline phosphatase (ALP)-induced in situ generation of Prussian blue nanoparticles for photothermometric ALP detection.